PROJECT SUMMARY: Rickettsial diseases caused by the Anaplasmataceae family pathogens of the genera Ehrlichia and Anaplasma remain a growing public health concern for over three decades and are also the second leading cause of tick- borne human infections in the USA and many parts of the world. Despite the complex cellular environments of arthropods and vertebrates having sophisticated systems of defense, the Anaplasmataceae pathogens evolved strategies to evade host clearance. Our prior studies demonstrated that the differential expression in vertebrate and tick cells alters host responses leading to variations in vertebrate host clearance/persistence. The central hypothesis of our competing renewal application remains that E. chaffeensis differentially regulates its gene expression in response to host cell environmental signals and that the host cell-specific gene expression is essential for pathogen?s continued survival in vertebrate and tick cell environments. We have made substantial progress in addressing the three specific aims of the previous funded project. The progress from the prior funded cycle included numerous peer-reviewed publications, which paved the way for additional exciting new directions for this proposed competing renewal application. Goals set for the three new specific aims of this renewal application have a strong premise, as supported with published research results and with additional unpublished preliminary data. The extensive novel data generated with the previous R01 grant support formed a strong foundation for the following proposed three specific aims of this renewal application. 1) Characterize sequence-specific DNA binding proteins (DBPs) in E. chaffeensis impacting RNA polymerase function in support of understanding the pathogen?s host- specific differential gene expression. 2) Characterize the functional significance of genes identified as essential for E. chaffeensis in vivo growth. 3) Mutagenesis and in vivo screening in defining genomic regions critical for the bacterial pathogenesis in vertebrate and tick hosts. The proposed specific aims are a logical extension of the substantial progress we have made during the previous funding cycle. The project goals have a strong scientific premise, as they are supported with the research data reported in many peer-reviewed publications and with the inclusion of additional preliminary data.